Shapulatov Umidjon, van Hoogdalem Mark, Schreuder Marielle, Bouwmeester Harro, Abdurakhmonov Ibrokhim Y, van der Krol Alexander R
1Laboratory of Plant Physiology, Wageningen University, Droevendaalsesteeg 1, 6708 PD Wageningen, The Netherlands.
2Center of Genomics and Bioinformatics, Academy of Sciences of Uzbekistan, University Street-2, Qibray Region, Tashkent, Uzbekistan 111215.
Plant Methods. 2018 Sep 24;14:83. doi: 10.1186/s13007-018-0351-2. eCollection 2018.
Recently, putative pre-miRNAs locations have been identified in the introns of plant genes, raising the question whether such genes can show a dual functionality by having both correct maturation of the host gene pre-mRNA and maturation of the miRNAs from the intron. Here, we demonstrated that such dual functionality is indeed possible, using as host gene the firefly luciferase gene with intron (ffgLUC), and different artificial intronic miRNAs (aimiRNA) placed within the intron of ffgLUC.
The miRNAs were based on the structure of the natural miR319a. Luciferase (LUC) activity in planta was used to evaluate a correct splicing of the ffgLUC mRNA. Different target sequences were inserted into the aimiRNA to monitor efficiency of silencing of different target mRNAs. After adjusting the insertion cloning strategy, the ffgLUC gene showed dual functionality with correct splicing of ffgLUC and efficient silencing of TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1 transcription factor genes targeted in-trans by aimiR-319a or targeting the transgene ffLUC in-cis by an aimiR-LUC. Silencing of endogenous target genes by aimiRNA or amiRNA is efficient both in transient assays and stable transformants. A behave as strong phenotype the PHYTOCHROME B (PHYB) gene was also targeted by ffgLUC. The lack of silencing of the PHYB target was most likely due to an insensitive target site within the PHYB mRNA which can potentially form a double stranded stem structure.
The combination of an overexpression construct with an artificial intronic microRNA allows for a simultaneous dual function in plants. The concept therefore adds new options to engineering of plant traits that require multiple gene manipulations.
最近，在植物基因的内含子中发现了假定的前体miRNA位置，这引发了一个问题，即这些基因是否可以通过使宿主基因前体mRNA正确成熟以及内含子中的miRNA成熟而具有双重功能。在这里，我们使用带有内含子的萤火虫荧光素酶基因（ffgLUC）作为宿主基因，并在ffgLUC的内含子中放置不同的人工内含子miRNA（aimiRNA），证明了这种双重功能确实是可能的。
这些miRNA基于天然miR319a的结构。利用植物中的荧光素酶（LUC）活性来评估ffgLUC mRNA的正确剪接。将不同的靶序列插入aimiRNA中，以监测不同靶mRNA的沉默效率。调整插入克隆策略后，ffgLUC基因显示出双重功能，ffgLUC正确剪接，并且aimiR - 319a在反式中靶向的TEOSINTE BRANCHED1/CYCLOIDEA/PROLIFERATING CELL FACTOR1转录因子基因或aimiR - LUC在顺式中靶向转基因ffLUC的沉默效率很高。在瞬时测定和稳定转化体中，aimiRNA或amiRNA对内源靶基因的沉默都是有效的。ffgLUC还靶向了表现出强表型的植物色素B（PHYB）基因。PHYB靶标的沉默缺失最可能是由于PHYB mRNA内的一个不敏感靶位点，该位点可能形成双链茎结构。
过表达构建体与人工内含子微小RNA的组合允许在植物中同时具有双重功能。因此，这一概念为需要多种基因操作的植物性状工程增加了新的选择。
